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1.
The ATP/ADP exchange is shown to be a partial reaction of the by the absence of measurable nucleoside diphosphokinase activity and the insensitivity of the reaction to , -di(adenosine-5′) pentaphosphate, a myokinase inhibitor. The exchange demonstrates an absolute requirement for Mg2+ and is optimal at an ADP/ATP ratio of 2. The high ATP concentration () required for maximal exchange is interpreted as evidence for the involvement of a low affinity form of nucleotide site. The ATP/ADP exchange is regarded as evidence for an ADP-sensitive form of the phosphoenzyme. In native enzyme, pre-steady state kinetics show that the formation of the phosphoenzyme is partially sensitive to ADP while modification of the enzyme by pretreatment with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of Mg2+ results in a steady-state phosphoenzyme population, a component of which is ADP sensitive. The ATP/ADP exchange reaction can be either stimulated or inhibited by the presence of K+ as a function of pH and Mg2+. 相似文献
2.
Purified cytochrome from bovine adrenocortical mitochondria was incorporated into liposomes by the cholate-dilution method utilizing either dialysis or Sephadex gel filtration. Among synthetic phospholipids tested, dioleoylglycerophosphocholine showed the best stability during the incorporation of into liposomes. A maximum amount of heme was incorporated into liposomes at a molar ratio of phospholipid to the cytochrome of approx. 200. When was incorporated into the dioleoylglycerophosphocholine liposomes by the cholate-filtration method, the showed two major populations on the elution pattern of the Sepharose 4B gel filtration, and were seen at a diameter of 200–600 Å and its aggregated forms. When the cytochrome was incorporated into dioleoylglycerophosphocholine liposomes or cholesterol-free adrenocortical mitochondrial liposomes, was less stable than in aqueous solution. Cholesterol or adrenodoxin markedly stabilized the liposomal . Liposomal required cholesterol for its optimum reduction with adrenodoxin, adrenodoxin reductase, and NADPH in the presence of CO. About 70% of the total heme in the dioleoylglycerophosphocholine liposomes was reduced by the enzymatic reduction in the presence of cholesterol, indicating that 70% of the total molecules are exposed to the surface of the outer monolayer. In order to see the location of the heme in membrane, the dioleoylglycerophosphocholine-liposomal was subjected to treatment. This reagent destroyed the liposomal . These results suggest that the heme is located in the proximity of the reacting sites which are exposed to the surface, or located on the vincinity of polar heads of the membrane. 相似文献
3.
The partial purification of from pig lens has been achieved by treatment with deoxycholate followed by density gradient centrifugation. The specific activity of the final preparation, ranging from 300 to 500 nmol/h per mg protein, is increased approx. 100-fold compared to the homogenate. A parallel increase in activity is also observed. Sodium dodecyl sulfate (SDS) gel electrophoresis reveals six major protein bands, one of which is the 93 kDa α subunit of which can be phosphorylated by reaction with [γ-32P]ATP. A second band contains a glycoprotein which displays an apparent molecular weight of 51 000 and thus appears to be the β subunit of the enzyme. The enzyme is sensitive to ouabain with the for and inhibition being 1.2 and 1.3 μM, respectively. Several agents which inhibit from other tissues such as oligomycin, Ca2+, vanadate, , (PCMBS) and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) also inhibit the lens enzyme. Monovalent cations other than K+ are partially effective in activating the ()-ATPase and activities. The K+ congeners were relatively more effective in supporting compared to activity. Other kinetic properties of the lens enzyme are also comparable to those of the enzyme from other tissues. Utilizing the partially purified membrane bound enzyme, discontinuities in Arrhenius plots of activity, activity and fluoresence polarization of the fluidity probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), are observed near the physiological temperature of lens. The possible significance of these observations for the mechanism of cataract formation are discussed. 相似文献
4.
Showdomycin inhibited pig brain ( with pseudo first-order kinetics. The rate of inhibition by showdomycin was examined in the presence of 16 combinations of four ligands, i.e., Na+, K+, Mg2+ and ATP, and was found to depend on the ligands added. Combinations of ligands were divided into five groups in terms of the magnitude of the rate constant; in the order of decreasing rate constants these were: (1), (2) , , and none, (3) , and , (4) and , , , and ATP. The highest rate was obtained in the presence of Na+, Mg2+ and ATP. The apparent concentrations of Na+, Mg2+ and ATP for half-maximum stimulation of inhibition () were 3 mM, 0.13 mM and 4μM, respectively. The rate was unchanged upon further increase in Na+ concentration from 140 to 1000 mM. The rates of inhibition could be explained on the basis of the enzyme forms present, including E1, E2, ES, E1-P and E2-P, i.e., E2 has higher reactivity with showdomycin than E1, while E2-P has almost the same reactivity as E1-P. We conclude that the reaction of ( proceeds via at least four kinds of enzyme form (E1, E2, E1 · nucleotide and EP), which all have different conformations. 相似文献
5.
Background
Assignment of function to new molecular sequence data is an essential step in genomics projects. The usual process involves similarity searches of a given sequence against one or more databases, an arduous process for large datasets. 相似文献6.
The modulating effect of membrane lipids on enzyme function has been described by several investigators. We have used the spin probe methyl stearate (M 12-NSE) to study this interaction in ox brain membranes enriched with . This methyl ester of stearic acid is practically insoluble in aqueous media, and consequently spectra of M 12-NSE-labelled preparations are free of “liquid lines”.At least two types of spectra may be obtained when ox brain microsomes are spin labelled with M 12-NSE, indicating the presence of two distinct binding sites. At one site the spin label is relatively unrestricted and gives rise to an isotropic spectrum. A second spectrum, which is obtained from spin label at another site, is similar to that which is observed after incorporation of M 12-NSE into phospholipid bilayers. This suggests that this latter site is within the core of the microsomal membrane.The two binding sites differ in their affinity for the spin probe. The low affinity site is both more abundant in crude preparations and is more easily removed by detergent treatment; spin labels at this site produce isotropic spectra. The high affinity sites are fewer in number and produce broad spectra. In addition these high affinity sites increase in concentration as the enzyme undergoes purification.The two sites are quite distinct in their sensitivity to ascorbic acid, the low affinity site showing a considerably greater rate of reduction by this agent.This study also demonstrates that the delipidation effects of sodium dodecyl sulfate and sodium deoxycholate on microsomes from ox brain are not identical.It is suggested that the two spin probe binding sites represent two different lipid domains, one of which is very closely associated with the enzyme and may reflect a protein-directed phospholipid specificity for this enzyme. 相似文献
7.
目的甲型H1N1流感病毒A/California/7/2009与A/California/4/2009病毒序列比较同源性在99%以上,本实验旨在比较两株病毒感染BALB/c小鼠研究感染力强弱。方法分别将A/California/7/2009(CA7)与A/California/4/2009(CA4)两株病毒分别连续10倍稀释后,对4~6周龄雌性BALB/c小鼠经乙醚麻醉后进行滴鼻攻毒,每个稀释度接种10只实验小鼠,测定CA7 MLD50为101.24/0.05 mL,检测小鼠感染、致病的多项指标,观察期为14 d。结果相同TCID50的CA7和CA4病毒感染小鼠,CA4感染小鼠后14 d内死亡率为20%,而CA7感染小鼠后8 d内死亡率为100%。CA7 106TCID50感染的小鼠病理表现为重度弥漫性间质性肺炎,CA4 106TCID50感染的小鼠病理表现为中度-重度间质性肺炎。结论在相同条件下,CA7感染力明显强于CA4。 相似文献
8.
Flaviano S. Martins Aparecida A. Silva Angélica T. Vieira Flávio H. F. Barbosa Rosa M. E. Arantes Mauro M. Teixeira Jacques Robert Nicoli 《Archives of microbiology》2009,191(8):623-630
The present work investigates some probiotic properties of four different microorganisms (Bifidobacterium animalis var. lactis BB-12, Escherichia coli EMO, Lactobacillus casei and Saccharomyces boulardii). In vitro and in vivo tests were carried out to compare cell wall hydrophobicity, production of antagonistic substances,
survival capacity in the gastrointestinal tract of germ-free mice without pathological consequence, and immune modulation
by stimulation of Küpffer cells, intestinal sIgA and IL-10 levels. In vitro antagonism against pathogenic bacteria and yeast
was only observed for the probiotic bacteria B. animalis and L. casei. The hydrophobic property of the cell wall was higher for B. animalis and E. coli EMO, and this property could be responsible for a better ability to colonize the gastrointestinal tract of germ-free mice.
Higher levels of sIgA were observed mainly for S. boulardii, followed by E. coli EMO and B. animalis, and only S. boulardii induced a significant higher level of IL-10. In conclusion, for a probiotic use, S. boulardii presented better characteristics in terms of immunomodulation, and B. animalis and L. casei for antagonistic substance production. The knowledge of the different probiotic properties could be used to choice the better
microorganism depending on the therapeutic or prophylactic application. 相似文献
9.
A.C. Simmonds J.M. East O.T. Jones E.K. Rooney J. McWhirter A.G. Lee 《生物化学与生物物理学报:生物膜》1982,693(2):398-406
Quenching of the fluorescence of the purified from muscle sarcoplasmic reticulum can be used to measure relative binding constants of hydrophobic compounds to the phospholipid-protein interface. We show that the binding constant for cholesterol is considerably less than that for phosphatidylcholine, so that cholesterol is effectively excluded from the phospholipid annulus around the ATPase. However, dibromocholestan-3β-ol causes quenching of the fluorescence of the ATPase, and so has access to other, non-annular sites. We suggest that these non-annular sites could be at protein/protein interfaces in ATPase oligomers. Oleic acid can bind at the phospholipid/protein interface, although its binding constant is less than that for a phosphatidylcholine, and it can also bind at the postulated non-annular sites. The effects of these compounds on the activity of the ATPase depend on the structure of the phospholipid present in the systems. 相似文献
10.
The parameters estimated from traditional A/C
i curve analysis are dependent upon some underlying assumptions that substomatal CO2 concentration (C
i) equals the chloroplast CO2 concentration (C
c) and the C
i value at which the A/C
i curve switches between Rubisco- and electron transport-limited portions of the curve (C
i-t) is set to a constant. However, the assumptions reduced the accuracy of parameter estimation significantly without taking
the influence of C
i-t value and mesophyll conductance (g
m) on parameters into account. Based on the analysis of Larix gmelinii’s A/C
i curves, it showed the C
i-t value varied significantly, ranging from 24 Pa to 72 Pa and averaging 38 Pa. t-test demonstrated there were significant differences in parameters respectively estimated from A/C
i and A/C
c curve analysis (p<0.01). Compared with the maximum ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) carboxylation rate (Vcmax), the maximum electron transport rate (Jmax) and Jmax/Vcmax estimated from A/C
c curve analysis which considers the effects of g
m limit and simultaneously fits parameters with the whole A/C
c curve, mean Vcmax estimated from A/C
i curve analysis (Vcmax-C
i) was underestimated by 37.49%; mean Jmax estimated from A/C
i curve analysis (Jmax-C
i) was overestimated by 17.8% and (Jmax-C
i)/(Vcmax-C
i) was overestimated by 24.2%. However, there was a significant linear relationship between Vcmax estimated from A/C
i curve analysis and Vcmax estimated from A/C
c curve analysis, so was it Jmax (p<0.05). 相似文献
11.
The interaction between the and the adenylate cyclase enzyme systems was examined. Cyclic AMP, but not 5′-AMP, cyclic GMP or 5′-GMP, could inhibit the enzyme present in crude rat brain plasma membranes. On the other hand, the cyclic AMP inhibition could not be observed with purified preparations of enzyme. Rat brain synaptosomal membranes were prepared and treated with either NaCl or cyclic AMP plus NaCl as described by Corbin, J., Sugden, P., Lincoln, T. and Keely, S. ((1977) J. Biol. Chem. 252, 3854–3861). This resulted in the dissociation and removal of the catalytic subunit of a membrane-bound cyclic AMP-dependent protein kinase. The decrease in cyclic AMP-dependent protein kinase activity was accompanied by an increase in activity. Exposure of synaptosomal membranes containing the cyclic AMP-dependent protein kinase holoenzyme to a specific cyclic AMP-dependent protein kinase inhibitor resulted in an increase in enzyme activity. Synaptosomal membranes lacking the catalytic subunit of the cyclic-AMP-dependent protein kinase did not show this effect. Reconstitution of the solubilized membrane-bound cyclic AMP-dependent protein kinase, in the presence of a neuronal membrane substrate protein for the activated protein kinase, with a purified preparation of , resulted in a decrease in overall activity in the presence of cyclic AMP. Reconstitution of the protein kinase alone or the substrate protein alone, with the has no effect on activity in the absence or presence of cyclic AMP. Preliminary experiments indicate that, when the activated protein kinase and the substrate protein were reconstituted with the enzyme, there appeared to be a decrease in the Na+-dependent phosphorylation of the Na+-ATPase enzyme, while the K+-dependent dephosphorylation of the was unaffected. 相似文献
12.
Tadashi Ogawa Mitsuru Fukuda Kei Sasaoka 《Biochimica et Biophysica Acta (BBA)/General Subjects》1973,297(1):60-69
One of the ninhydrin-negative alanine conjugates isolated from pea seedlings was identified as .The identification of this conjugate was carried out by a comparison of its gas-liquid chromatographic and mass spectrometric properties, and its nuclear magnetic resonance and infrared spectra with those of synthetic . The alanine in the conjugate was shown to be present as the D-isomer by enzymatic and chromatographic analyses. 相似文献
13.
Kristin K Nicodemus 《BMC bioinformatics》2008,9(1):130
Background
Risk for complex disease is thought to be controlled by multiple genetic risk factors, each with small individual effects. Meta-analyses of several independent studies may be helpful to increase the ability to detect association when effect sizes are modest. Although many software options are available for meta-analysis of genetic case-control data, no currently available software implements the method described by Kazeem and Farrall (2005), which combines data from independent family-based and case-control studies. 相似文献14.
Structural changes in the purified accompanying detergent inactivation were investigated by monitoring changes in light scattering, intrinsic protein fluorescence, and tryptophan to β-parinaric acid fluorescence resonance energy transfer. Two phases of inactivation were observed using the non-ionic detergents, digitonin, Lubrol WX and Triton X-100. The rapid phase involves detergent monomer insertion but little change in protein structure or little displacement of closely associated lipids as judged by intrinsic protein fluorescence and fluorescence resonance energy transfer. Lubrol WX and Triton X-100 also caused membrane fragmentation during the rapid phase. The slower phase of inactivation results in a completely inactive enzyme in a particle of 400 000 daltons with 20 mol/mol of associated phospholipid. Fluorescence changes during the course of the slow phase indicate some dissociation of protein-associated lipids and an accompanying protein conformational change. It is concluded that non-parallel inhibition of and activity by digitonin (which occurs during the rapid phase of inactivation) is unlikely to require a change in the oligomeric state of the enzyme. It is also concluded that at least 20 mol/mol of tightly associated lipid are necessary for either or activity and that the rate-limiting step in the slow inactivation phase involves dissociation of an essential lipid. 相似文献
15.
(1) Eosin bound to the in the presence of K+ has practically the same fluorescence as eosin without enzyme while in the presence of Na+ the fluorescence is higher, the excitation maximum is shifted from 518 to 524 nm, the emission maximum from 538 to 542 nm, and a shoulder appears at about 490 nm on the excitation curve. (2) The amount of eosin bound increases with the K+ concentration but with a low affinity. With equal concentrations of Na+ and K+ more is bound in the presence of Na+, and the difference between 150 mM Na+ and 150 mM K+ shows one high-affinity eosin binding site per 32P-labelling site ( 0.45 μM). With lower concentrations of the cations there are between one and two Na+-dependent high-affinity eosin binding sites per 32P-labelling site. (3) ATP (and ADP) prevents the hig-affinity Na+-dependent eosin binding and there is competition between eosin and ATP for the hydrolysis in the presence of Na+ (+Mg2+). (4) Eosin, like ATP, increases the Na+ relative to K+ affinity ) for Na+ activation of hydrolysis and for Na+ protection against inactivation by . (5) The results suggest that the high affinity eosin binding site is an ATP binding site and that it is located on the enzyme in an environment with a low polarity, i.e., the conformational change induced by Na+ opens a high-affinity site for ATP while K+ closes the site (or decreases the affinity to a low level). The experiments suggest, furthermore, that the ATP which increases the Na+ relative to K+ affinity of the internal sites is not the ATP which is hydrolyzed, i.e., in a turnover cycle in the presence of the system reacts with two different ATP molecules. 相似文献
16.
Although acute alterations in Ca2+ fluxes may mediate the skeletal responses to certain humoral agents, the processes subserving those fluxes are not well understood. We have sought evidence for Ca2+-dependent ATPase activity in isolated osteoblast-like cells maintained in primary culture. Two Ca2+-dependent ATPase components were found in a plasma membrane fraction: a high affinity component (half-saturation constant for Ca2+ of 280 nM, of 13.5 nmol/mg per min) and a low affinity component, which was in reality a divalent cation ATPase, since Mg2+ could replace Ca2+ without loss of activity. The high affinity component exhibited a pH optimum of 7.2 and required Mg2+ for full activity. It was unaffected by potassium or sodium chloride, ouabain or sodium azide, but was inhibited by lanthanum and by the calmodulin antagonist trifluoperazine. This component was prevalent in a subcellular fraction which was also enriched in 5′-nucleotidase and adenylate cyclase activities, suggesting the plasma membrane as its principal location. Osteosarcoma cells, known to resemble osteoblasts in their biological characteristics and responses to bone-seeking hormones, contained similar ATPase activities. Inclusion of purified calmodulin in the assay system caused small non-reproducible increases in the Ca2+-dependent ATPase activity of EGTA-washed membranes. Marked, consistent calmodulin stimulation was demonstrated in membranes exposed previously to trifluoperazine and then washed in trifluoperazine-free buffer. These results indicate the presence of a high affinity, calmodulin-sensitive Ca2+-dependent ATPase in osteoblast-like bone cells. As one determinant of Ca2+ fluxes in bone cells, this enzyme may participate in the hormonal regulation of bone cell function. 相似文献
17.
(1) ( from rectal glands of the spiny dogfish has been reconstituted into phospholipid vesicles. The nonionic detergent octaethyleneglycoldodecyl monoether (C12E8) is used to dissolve both the enzyme and the lipids and reconstitution is accomplished by subsequent removal of the detergent by adsorption to polystyrene beads. (2) About 60% of the enzyme incorporates in the right-side-out orientation (r/o). The fraction of molecules in the inside-out orientation (i/o) increases from about 10% to about 30% with a parallel decrease in the fraction of ‘non-oriented’ (n-o) molecules (both sides exposed) when the protein/lipid ratio decreases from 1:10 to 1:75. (3) The orientation of enzyme molecules detected from vanadate binding is the same as measured from activity, i.e., the turnover of the enzyme molecule in the diffrent orientations is the same. (4) The recovery of the specific activity of the incorporated enzyme increases with an increase in the protein/lipid ratio and is 100% with a protein/lipid ration of about 1:20 or higher. Full recovery is only obtained provided a proper lipid composition is chosen which includes both negatively charged phospholipids, preferably phosphatidylinositol, and cholesterol. (5) The ATP-dependent, K+-stimulated Na+-influx is found to be about 35 μmol Na+ per mg (i/o)-protein per min at 22°C in 1:10 protein/lipid liposomes. The specific activity corresponds to 3 Na+ transported per ATP molecule hydrolyzed. 相似文献
18.
Yoshio Ushijima Minoru Nakano 《Biochemical and biophysical research communications》1980,93(4):1232-1237
The HOCl in chlorine-water oxidizes DPF to cis-DBE in parallel to the HOCl concentration. The addition of H2O2 produces singlet molecular oxygen, and a bimol collision above pH 6.0, but not in the pH region 3.0 to 4.0. The DPF conversion to cis-DBE is initiated by a 1,2-position attack of OH? and Cl+, followed by the HCl elimination. The oxidation potency of HOCl is much greater than the singlet molecular oxygen generated in chlorine-water/H2O2 solution, on the pH range 6.0 to 8.0 where both HOCl and OCl? are present. 相似文献
19.
20.
(1) A quantitative study has been made of the binding of ouabain to the ( in homogenates prepared from brain tissue of the hawk moth, Manduca sexta. The results have been compared to those obtained in bovine brain microsomes. (2) The insect brain ( will bind ouabain either in the presence of Mg2+ and Pi, (‘Mg2+, Pi’ conditions) or in the presence of Na+, Mg2+, and an adenine nucleotide (‘nucleotide’ conditions) as is the case for the bovine brain (. The binding conditions did not alter the total number of receptor sites measured at high ouabain concentrations in either tissue. (3) Potassium ion decreases the affinity (increases the ) of ouabain to the M. sexta brain ( under both binding conditions. However, ouabain binding is more sensitive to K+ inhibition under the nucleotide conditions. In bovine brain ouabain binding is equally sensitive to K+ inhibition under the both conditions. (4) The enzyme-ouabain complex has a rate of dissociation that is 10-fold faster in the M. sexta preparation than in the bovine brain preparation. Because of this, the M. sexta ( has a higher for ouabain binding and is less sensitive to inhibition by ouabain than the bovine brain enzyme. (5) This data supports the hypothesis that two different conformational states of the M. sexta ( can bind ouabain. 相似文献